1. Field of the Invention
The present invention involves processes for 1,2-dehydrogenation of certain .DELTA..sup.4 -3-keto C.sub.21 -hemiester steroids with Arthrobacter simplex or Bacterium cyclooxydans to produce the corresponding .DELTA..sup.1,4 -3-keto C.sub.21 hemiester or free alcohol with improved yields, and shorter bioconversion time.
2. Description of the Related Art
The 1,2-dehydrogenation of .DELTA..sup.4 -3-keto steroids to the corresponding .DELTA..sup.1,4 -3-keto steroids with Septomyxa affinis, A. simplex and other microorganisms is well known in the art and has valuable commercial significance.
U.S. Pat. No. 2,837,464 claims a process for 1,2-dehydrogenation of .DELTA..sup.4 -3-ketopregnenes to .DELTA..sup.1,4 -pregnadienes where the C.sub.21 hydroxyl group is either free or esterified with a monocarboxylic acid by the use of Corynebacterium (Arthrobacter) simplex or Corynebacterium hoagii.
U.S. Pat. Nos. 2,902,410 and 2,902,411 claim processes for the 1,2-dehydrogenation of 1,2-saturated steroids to 1,2-dehydro steroids with Septomyxa affinis.
U.S. Pat. No. 3,360,439 discloses a process for the 1,2-dehydrogenation of .DELTA..sup.4 -3-keto steroids to the corresponding .DELTA..sup.1,4 -3-keto steroids using A. simplex cells which had been previously treated with a lower alkanol or alkanone. The process of the present invention does not require pretreatment of the cells and may be performed using viable cells in a fermentation broth. Alternatively cells pretreated by air or heat drying or by exposure to a lower alkanone such as acetone may be used. The steroid-1-dehydrogenase of these organisms can also be used in a cell-free form.
U.S. Pat. No. 3,770,586 claims an improved process for the production of 11.beta.,21-dihydroxypregna-1,4,17(20)-trien-3-one (Example 1) and 11.beta.,21-dihydroxy-6.alpha.-methylpregna-1,4,17(20)-trien-3-one (Example 5) whereby the alkali metal salts of the .DELTA..sup.4 -3-keto 21-hemiester substrates thereof where the acyl radical is that of a dicarboxylic acid of 3 to 12 carbon atoms are 1,2-dehydrogenated by use of S. affinis. Using shake flasks, 11.beta.,21-dihydroxypregna-4,17(20)-dien-3-one 21-hemisuccinate potassium salt at a concentration of 5.0 g/l and medium 1, the desired .DELTA..sup.1,4 -product was produced in 100% yield after 5 days (Example 8). Example 1 uses larger stirred tanks and shows a lower yield. With medium 2, and 2.0-7.0 g/l of 11.beta.,21-dihydroxypregna-4,17(20)-dien-3-one 21-hemisuccinate potassium salt yields of 91.1-96.6% were obtained in 6 days.
U.S. Pat. Nos. 4,524,134 and 4,704,358 disclose improved microbial processes to produce a 1,2-dehydrogenated steroid from the corresponding 1,2-saturated .DELTA..sup.4 -3-keto-steroid using air-dried or heat-dried cells of Arthrobacter simplex and Bacterium cyclooxydans respectively.
The processes of the present invention provide improved yields and at the same time permit the use of higher substrate concentrations and reduced reaction times.